The first of two developmental switches in human hemoglobin expression involves the down-regulation or silencing of the embryonic epsilon-globin gene. We have previously reported a silencer element (epsilon GS) in the 5' flanking region located at -270 bp 5' to the cap site of the epsilon-globin gene that provides negative regulation for this autonomously regulated gene. We now have extended this analysis 5' as far as to the globin locus control region (LCR) and have identified several functionally important cis-elements that markedly affect the epsilon-globin promoter. We use reporter gene constructs with deletion mutations in the 5' epsilon-globin DNA and transient transfection and DNA-protein binding assays. A series of negative and positive elements are present along the 6 kb of 5' epsilon-promoter, including two strong negative control elements located at -3 kb and -1.7 kb, designated as epsilon-NRA and epsilon-NRB, respectively. In addition, we have identified several positive elements in this sequence. These data suggest the complexity of globin switching, in this case the down regulation of epsilon-globin gene. Several DNA motifs for the erythroid specific transcription factor GATA-1 within the negative control element epsilon-NRA have been identified and characterized. The study suggests that besides epsilon-GS, the epsilon-globin gene switch-off requires the combined actions of these multiple negative elements. Identifying the cis-acting regulating elements in the 5' region of the epsilon-globin gene and elucidating the mechanism of epsilon-globin gene silencing may provide an understanding about the coordinated regulation of the entire beta-globin cluster. This project has been combined with